scholarly journals Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ28 RNA Polymerase

2006 ◽  
Vol 188 (15) ◽  
pp. 5524-5531 ◽  
Author(s):  
Hilda Hiu Yin Yu ◽  
Elizabeth G. Di Russo ◽  
Megan A. Rounds ◽  
Ming Tan
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
Promoter Sequence ◽  
Promoter Element ◽  
Developmental Gene ◽  
Spacer Length ◽  
E Coli ◽  
Promoter Recognition ◽  
Developmental Gene Regulation

ABSTRACT σ28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial σ28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the σ28-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a −35 element recognized by chlamydial σ28 RNA polymerase that resembles the consensus −35 sequence. Within the −10 element, however, chlamydial σ28 RNA polymerase showed a striking preference for a CGA sequence at positions −12 to −10 rather than the longer consensus −10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli σ28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the −10 promoter element recognized by σ28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that σ28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for σ28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred σ28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n11/12-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n11/12 is a spacer of 11 or 12 nt.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

scholarly journals DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS

1984 ◽  
Vol 224 (3) ◽  
pp. 799-815 ◽  
Author(s):  
J E Walker ◽  
N J Gay ◽  
M Saraste ◽  
A N Eberle
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Gene Encoding ◽  
E Coli ◽  
Extensive Region ◽  
Transcriptional Promoters ◽  
Phosphoribosylpyrophosphate Amidotransferase ◽  
Unc Operon

The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS. Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E. coli genome. Sequences that are probably transcriptional promoters for unc and phoS can be detected and the identity of the unc promoter has been confirmed by experiments in vitro with RNA polymerase. Upstream of the promoter sequence is an extensive region that appears to be non-coding. Conserved sequences are found that may serve to concentrate RNA polymerase in the vicinity of the unc promoter. Hairpin loop structures resembling known rho-independent transcription termination signals are evident following the unc operon and glmS. The glmS gene encoding the amidotransferase, glucosamine synthetase, has been identified by homology with glutamine 5-phosphoribosylpyrophosphate amidotransferase.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

scholarly journals Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain

2001 ◽  
Vol 183 (2) ◽  
pp. 570-579 ◽  
Author(s):  
Michal Gropp ◽  
Yael Strausz ◽  
Miriam Gross ◽  
Gad Glaser
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Amino Acid Starvation ◽  
E Coli ◽  
Terminal Domain ◽  
Negative Effect ◽  
And Control ◽  
Two Hybrid

ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

scholarly journals Biophysical Properties of Escherichia coli Cytoplasm in Stationary Phase by Superresolution Fluorescence Microscopy

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Yanyu Zhu ◽  
Mainak Mustafi ◽  
James C. Weisshaar
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Mean Square Displacement ◽  
Quiescent State ◽  
Chromosomal Dna ◽  
Content Type ◽  
E Coli

ABSTRACT In nature, bacteria must survive long periods of nutrient deprivation while maintaining the ability to recover and grow when conditions improve. This quiescent state is called stationary phase. The biochemistry of Escherichia coli in stationary phase is reasonably well understood. Much less is known about the biophysical state of the cytoplasm. Earlier studies of harvested nucleoids concluded that the stationary-phase nucleoid is “compacted” or “supercompacted,” and there are suggestions that the cytoplasm is “glass-like.” Nevertheless, stationary-phase bacteria support active transcription and translation. Here, we present results of a quantitative superresolution fluorescence study comparing the spatial distributions and diffusive properties of key components of the transcription-translation machinery in intact E. coli cells that were either maintained in 2-day stationary phase or undergoing moderately fast exponential growth. Stationary-phase cells are shorter and exhibit strong heterogeneity in cell length, nucleoid volume, and biopolymer diffusive properties. As in exponential growth, the nucleoid and ribosomes are strongly segregated. The chromosomal DNA is locally more rigid in stationary phase. The population-weighted average of diffusion coefficients estimated from mean-square displacement plots is 2-fold higher in stationary phase for both RNA polymerase (RNAP) and ribosomal species. The average DNA density is roughly twice as high as that in cells undergoing slow exponential growth. The data indicate that the stationary-phase nucleoid is permeable to RNAP and suggest that it is permeable to ribosomal subunits. There appears to be no need to postulate migration of actively transcribed genes to the nucleoid periphery. IMPORTANCE Bacteria in nature usually lack sufficient nutrients to enable growth and replication. Such starved bacteria adapt into a quiescent state known as the stationary phase. The chromosomal DNA is protected against oxidative damage, and ribosomes are stored in a dimeric structure impervious to digestion. Stationary-phase bacteria can recover and grow quickly when better nutrient conditions arise. The biochemistry of stationary-phase E. coli is reasonably well understood. Here, we present results from a study of the biophysical state of starved E. coli. Superresolution fluorescence microscopy enables high-resolution location and tracking of a DNA locus and of single copies of RNA polymerase (the transcription machine) and ribosomes (the translation machine) in intact E. coli cells maintained in stationary phase. Evidently, the chromosomal DNA remains sufficiently permeable to enable transcription and translation to occur. This description contrasts with the usual picture of a rigid stationary-phase cytoplasm with highly condensed DNA.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

Peptide nucleic acid restores colistin susceptibility through modulation of MCR-1 expression in Escherichia coli

2020 ◽  
Author(s):  
Xiaoming Wang ◽  
Yao Wang ◽  
Zhuoren Ling ◽  
Chaoyang Zhang ◽  
Mingming Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peptide Nucleic Acid ◽  
Promoter Activity ◽  
Sequence Variation ◽  
Promoter Sequence ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Expression Host ◽  
Antisense Approach ◽  
Increased Susceptibility

Abstract Background Plasmid-mediated mechanisms of drug resistance accelerate the spread of polymyxin resistance, leaving clinicians with few or no antibacterial options for the treatment of infections caused by MDR bacteria, especially carbapenemase-producing strains. Objectives To evaluate the associations among promoter sequence variation, mcr-1 expression, host factors and levels of colistin resistance and to propose antisense agents such as peptide nucleic acids (PNAs) targeting mcr-1 as a tool to restore colistin susceptibility through modulation of MCR-1 expression in Escherichia coli. Methods A β-galactosidase assay was performed to study mcr-1 promoter activity. Quantitative real-time PCR and western blot assays were used to identify the expression level of MCR-1 in WT strains and transformants. Three PNAs targeting different regions of mcr-1 were designed and synthesized to determine whether they can effectively inhibit MCR-1 expression. MIC was measured to test colistin susceptibility in the presence or absence of PNA-1 in mcr-1-carrying E. coli. Results Variation in the mcr-1 promoter sequence and host species affect promoter activity, MCR-1 expression levels and colistin MICs. One PNA targeting the ribosome-binding site fully inhibited the expression of mcr-1 at a concentration of 4 μM, resulting in significantly increased susceptibility to colistin. The MIC90 of colistin decreased from 8 to 2 mg/L (P < 0.05) in the presence of 4 μM PNA. Conclusions These findings suggest that the antisense approach is a possible strategy to combat mcr-1-mediated resistance as well as other causes of emerging global resistance.

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